Circadian rhythm is an intrinsic roughly-24-hour biological clock embedded within most living organisms. In mammals, circadian rhythm coordinates sleep-wake cycles, blood pressure, body temperature and liver metabolism in a daily cycle. In humans, long term disruption of circadian rhythm can impair physical and mental health. For instance, sleep disorders are circadian related and affect about 20% of Americans, resulting in higher healthcare costs and lost productivity. Developing new therapeutic agents against circadian related diseases requires a better understanding of the mechanism involved in regulating the circadian rhythm. It is widely accepted that the circadian rhythm is governed within the cell by a transcriptional-translational loop. In this loop, CLOCK (circadian locomotor output cycle kaput) and ARNTL (aryl hydrocarbon receptor nuclear translocator-like) form a heterodimer that binds to a DNA transcriptional element termed E-Box. The CLOCK/ARNTL/E-Box complex activates the transcription of circadian genes such as PER (period) and CRY (cryptochrome). After translating, PER and CRY down-regulate CLOCK/ARNTL's activity. This generates a time delayed negative feedback loop that sets the rhythmic expression of clock-related genes. Significant progress has been made towards understanding the basic biology of the circadian rhythm. However, the lack of structural studies of the key functional components of the circadian rhythm limits our understanding of the molecular mechanism of this important clockwork. In order to overcome this barrier, combinations of structural biochemical tools, traditional biochemical, and biophysical methods will be applied to investigate the interactions among core regulatory components in the mammalian circadian clockwork. The research will target two areas: (1) the CLOCK/ARNTL/E-Box complex that serves as the positive factor in the transcriptional- translational loop and (2) the negative feedback factors CRY and PER. In addition, the project will study the effect of an important metabolism cofactor, NAD (P) H, on the circadian complexes, which will link the cellular metabolism to the circadian rhythm. Mammalian CLOCK, ARNTL, PER and CRY will be expressed and their complexes will be in vitro assembled and analyzed using traditional biochemical and biophysical experiments such as electrophoresis, liquid chromatography, UV or fluorescent spectra, dynamic light scattering and surface plasmon resonance. Structures of individual proteins and protein/DNA complexes will be determined by X-ray crystallography, cryo-electron microscopy and small angle X-ray scattering. These structures will then be used to identify the key residues at the protein/DNA, protein/protein, and protein/ligand interface that are critical to the interaction between circadia core components. Conclusions regarding regulatory mechanisms of the circadian rhythm can be drawn from the various structures obtained, which will provide essential knowledge for future development of new therapeutic strategies against circadian disorders.